この作品は、自然科学とカナダ工学研究会議（助成＃249669および＃482986）とカナダの心臓と脳卒中財団（助成金＃485508）によって賄われています。著者は実験的なプロトコルを開発中で彼らの支援のためのユーセフ·エル·ハイエク博士とチーワンに感謝します。

動物の取り扱いに関係するすべての手順は、制度ガイドライン（プロトコルはありません。20009387）に従い、トロント動物ケア委員会の大学によって承認された。次のメソッドは、該当する場合、層流フードで、無菌のツールやテクニックを使用して実行されるべきである。 以下のプロトコルのテキストでは、フレーズ &quot;EFH-SFMは&quot;上皮細胞増殖因子、塩基性線維芽細胞増殖因子とヘパリンを補充無血清培地を指します。これらのマイトジェンは、未分化状態の4のNPCを維持するため、未分化NPCの走電性を調査するときEFH-SFMは使用されています。成熟細胞型に分化するように誘導NPCの走電性を調べるときは、 &quot;FBS-SFMは、&quot; 1％ウシ胎児血清を添加した無血清培地を指します。 FBSは、成熟した神経表現型5にNPCの分化を促進する。 1。神経前駆細胞の単離および培養（ない）映像に示すように<ol><li>頸椎脱臼経由イソフルランと犠牲とCD1マウス（6-8週齢）Anaesthetize。 </li><li> 70％エタノールでヘッドを消すと鋭い解剖ハサミで動物を斬る。 </li><li>外科手術用鉗子で頭を押さえながら、頭蓋骨を露出するために背側表面に皮膚を削除します。 </li><li>メスとNOを使用。 11ブレードは、中外軸に沿って、またrostrocaudal方向に矢状縫合に沿って前頭洞で頭蓋骨を獲得。 </li><li>離れていないと頭から頭頂骨をはがします。 7曲がった鉗子ではなく、ピアス脳組織に世話をして。 </li><li>小脳の下から開始し、嗅球に向かって進ん脳の下の薄いへらを挿入します。鉗子で適所に頭蓋骨を押さえながら、そっと頭蓋骨から脳を引くとすぐに氷のように冷たい人工脳脊髄液（以下のレシピを参照してください）​​に入れてください。 </li><li>解剖顕微鏡下では、無菌の使用解剖用はさみや鉗子は正中線に沿って半分に脳を切った。内側（カット）面が上方を向くようにそれぞれの半球を回転させます。 </li><li>半球を選択して、内側面を上に向けて、脳梁（脳梁の後部領域）の膨大部を見つけます。 </li><li>皮質の表面からの背腹軸に沿って脳梁膨大部への切り込みを入れる。 </li><li>側脳室の内側と外側の壁を露出させるために嗅球に向かって切開皮質ピール。 </li><li>背面が上方を向くように半球を回転させたり、切り取ってNPCが6に存在する脳室周囲白質領域が含まれている露出した内側と外側の壁を収集するために湾曲したmicroscissorsを使用しています。 </li><li>他の半球の手順1.8から1.11までを繰り返します。 </li><li> 15ccのチューブ内のトリプシン溶液（下記レシピを参照してください）​​の7ミリリットルに単離された組織をピペットで、37°C​​でロッカー上にチューブを配置25分間インキュベーター。 </li><li> 5分間1,500 rpmでチューブを遠心分離し、上清を吸引し、（下のレシピを参照してください）​​トリプシンインヒビター溶液2mlに組織を再懸濁する。 </li><li>ゆっくり気泡を避けるために慎重に小さなボーリング孔パスツールピペット30から50回で組織をひいて粉にする。 </li><li> 、5分間1,500 rpmでチューブを遠心し上清を吸引し、ペレットを3-5回摩砕することにより、SFM中1-2ミリリットル（下のレシピを参照してください）​​に再懸濁します。 </li><li> 3分間1,500 rpmでチューブを遠心分離し、SFMの1ミリリットル+ EFHで清と再懸濁を吸引除去する。 </li><li> SFM + EFHでμlあたり10細胞の密度でT25培養フラスコ内の血球とプレート細胞と生細胞密度をカウントします。 </li><li>文化はNPCに成る自由浮動プライマリーニューロスフィアを得るのに邪魔されずに7日間成長することができます。 </li></ol> 2。走電性商工会議所の準備<ol><li> NO 3正方形のガラスを置きます。 1カバースリップ（22×22×0.17ミリメートル）は、i一晩の6N塩酸Naのボトル。 </li><li>翌日には、正方形の無からのガラスの6短冊（22×5×0.17ミリメートル）をカットするダイヤモンドチップガラスカッターを使用しています。 1カバースリップ。 </li><li>層流フード内に酸洗浄した正方形と長方形のスリップスリップを転送します。組織培養グレードの水でオートクレーブした後、70％エタノールを持つ最初の長方形と正方形のストリップを洗い、拭いてキムに乾燥することができます（追加された不妊のため、ガラスは空気乾燥させることができる）。 </li><li>正方形のガラススライドの一方の面の周囲に真空グリースを塗布し、60ミリメートルプラスチックペトリ皿の底にそれらを封印。 </li><li>長方形のガラス片の一方の面の長軸に沿って、真空グリースを塗布し、中央の谷を作成するために正方形のガラススライドの反対側のエッジ（彼らが互いに平行となるように）にそれらを封印。 </li><li>層流フード内に少なくとも15分間のチャンバを紫外線殺菌。 </li><li>ポリ-L-lysiの250から300μlのピペット2時間室温でインキュベート室及び中央トラフの上にね。 </li><li>潜伏期間の終了前の約15分間は、（下のレシピを参照してください）​​マトリゲル溶液を調製します。 </li><li>ポリ-L-リジンを吸引除去し、オートクレーブ滅菌水1ml、中央谷の上にマトリゲル溶液をピペット250から300μlで中央の谷を洗います。 </li><li> 1時間37℃でチャンバーをインキュベートする。 </li><li>マトリゲルの液を除き、穏やかに、SFM中1-2 mlの中央谷を洗う。 </li><li>中央の谷へEFH-SFMまたはFBS-SFMの各々100μL添加し、カウントを顕微鏡のステージ上に走電性チャンバーを転送します。 </li><li>ピペット3月4日60ミリメートルペトリ皿に神経球含有培養mlのはカウント顕微鏡のステージにシャーレを転送します。 </li><li> 5倍の視聴目的で、それぞれの走電性の中心トラフに5月8日全神経球（最大4時）に転送するためにP10のピペットを使用それらを解離させずにチャンバー、慎重マトリゲル基板を中断せずに、中央の谷の周りの神経球を広げた。 </li><li>中央の谷にEFH-SFMまたはFBS-SFMの追加の150から200μlを添加する。 </li><li> 37℃、5％CO 2、17から20時間後には100％の加湿インキュベーターに走電性チャンバを転送（もし未分化のNPCを分析）を図1に示すように、ニューロスフィアは、マトリゲル基板に付着し、単一細胞に解離できるようにする。差別化されたNPCを分析する場合、潜伏期間は、細胞の分化を可能にするために69から72時間に延長すべきである。 </li></ol> 3。細胞タイムラプスイメージングを生きる<ol><li>ライブセルイメージングシステムは、37℃で平衡化させ℃、5％前タイムラプス撮影開始までの30分の最小のCO 2。 </li><li>一方の端部から直径1mmのシルバーワイヤー、コイルふたり12cmの部分をカットし、クロロックスbにそれらを配置する銀/塩化銀電極を形成するために、20分間浸出する。 </li><li>計数顕微鏡のステージ上に走電性室を移し、次の基準に基づいてライブセルイメージングの移行解析のために使用される室を選択します：i）ニューロスフィアは、単一細胞にほぼ完全に解離しなければならないと、ii）細胞が持っている必要があり少しツーないプロセスと丸い形態は、細胞体から伸びる。 </li><li>別の方形な​​いとともに、層流フードに選択された走電性チャンバーを転送します。 1ガラスカバースリップと真空グリース。 </li><li>オートクレーブした水で、次に70％エタノールを用いて第一滑り、カバースリップの2つの平行な辺に真空グリースのストリップを適用するカバーを洗う。 </li><li> 、室の中央トラフから培地を吸引除去した後すぐに2つの平行な矩形状のガラス片にグリスストリップの残りの部分は、効果的に屋根を作成するようなチャンバー上にカバースリップ（グリース側を下に向け）を配置室へ。 </li><li>毛細管現象を介して中央の谷に新鮮EFH-SFMまたはFBS-SFMの各々100μL添加します。 </li><li> 図2に示すように、中央の溝の両端に培地のプールの境界線を作成するために真空グリースを使用しています。 </li><li> PVCチューブの2 15cmの部分をカットし、丁寧に泡がチューブ内に形成されないの確保、チューブにアガロース溶液を注入する18ゲージの針で10ccのシリンジを使用し、ゲルを5分間凝固させる。 </li><li>アガロースゲルチューブ、銀/塩化銀電極、および培養培地リザーバーとして使用される空の60ミリメートルペトリ皿のペアと一緒に、ライブセルイメージングシステムへの走電性チャンバーを転送したAg / AgCl電極を含んでいます。走電性チャンバーは37内に物を置か℃、20〜30分間、5％CO 2環境。 </li><li>この時間の間に、ドリルで穴を開けることによって2つの空のペトリ皿のふたと走電性室のペトリ皿の蓋を準備それらの中に、図3に示すように、ドレメルまたは同様のツールを持つ。 </li><li>ピペット中央谷の両側の上EFH-SFMまたはFBS-SFMは1〜1.5 mlを加え、それぞれの空のシャーレにSFM 7-8 mlであった。各皿に走電性チャンバーの中央トラフと場所1 Ag / AgCl電極の両側に1つ配置シャーレ。 図4に示すように、アガロースゲルブリッジとの電気的導通を確立するために、走電性チャンバー、ペトリ皿とのギャップを埋める。 </li><li>電流を測定するために直列に電流計で、外部電源に銀/塩化銀電極を接続し、電源をオンにします。所望の電界強度が達成されるまで、直接中央谷を横切って電界強度を測定し、電源の出力を調整するために電圧計を使用します（このラボで行わアッセイはで250 mVの/ mmのdCEFの強みを活かし1と1.5ミリアンペア）の間に電流。 </li><li>イニシアTEは、タイムラプスライブセルイメージングシステム上のモジュール、および実験は、目的の時間のために実行することができます。検定終了後、標準の免疫染色解析のため、4％パラホルムアルデヒドで細胞を固定します。 </li></ol>

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特別な利害関係は宣言されません。

このプロトコルは、先行研究7-9のよく確立された方法の中から適応されています。 Galvanotactic室は、細胞播種の閉じ込めのためのよく独立したガラス張りの建設を含むさまざまなテクニック、のさまざまな方法を使って、または中央トラフ10,11の微細加工にCO 2レーザーアブレーション法を用いて構築することができる。いくつかのテクニックは他よりも面倒でコスト?...

<table border="1"><tbody><tr><td height="44"> アイテムの名前 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td></td><td></td><td></td><td> 神経前駆細胞の単離 </td></tr><tr><td> 2MのNaCl </td><td>シグマ</td><td> S5886 </td><td> 100ミリリットルのdH 2 Oに溶解11.688グラム</td></tr><tr><td> 1MのKCl </td><td>シグマ</td><td> P5405 </td><td> 100ミリリットルのdH 2 Oに溶解した7.456グラム</td></tr><tr><td> 1M MgCl 2を </td><td>シグマ</td><td> M2393 </td><td> 100ミリリットルのdH 2 Oに溶解20.33グラム</td></tr><tr><td> 155 MMのNaHCO 3 </td><td>シグマ</td><td> S5761 </td><td> 100ミリリットルのdH 2 Oに溶解した1.302グラム</td></tr><tr><td> 0.5Mグル​​コース</td><td>シグマ</td><td> G6152 </td><td> 100に溶解し9.01グラムミリリットルのdH 2 O </td></tr><tr><td> 108 mMのCaCl 2 </td><td>シグマ</td><td> C7902 </td><td> 100ミリリットルのdH 2 Oに溶解した1.59グラム</td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>ギブコ</td><td> 15070 </td><td></td></tr><tr><td>ウシ膵臓トリプシン</td><td>シグマ</td><td> T1005 </td><td></td></tr><tr><td>羊は精巣ヒアルロニダーゼ</td><td>シグマ</td><td> H6254 </td><td></td></tr><tr><td>キヌレン酸</td><td>シグマ</td><td> K3375 </td><td></td></tr><tr><td>オボムコイドトリプシンインヒビター</td><td>ワージントン</td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> 12100046 </td><td></td></tr><tr><td> [F12] </td><td>インビトロジェン</td><td> 21700075 </td><td></td></tr><tr><td> 30％のグルコース</td><td>シグマ</td><td> G6152 </td><td></td></tr><tr><td> 7.5パーセントのNaHCO 3 </td><td>シグマ</td><td> S5761 </td><td></td></tr><tr><td> 1M HEPES </td><td>シグマ</td><td> H3375 </td><td> 100ミリリットルのdH 2 Oに溶解23.83グラム</td></tr><tr><td> L-グルタミン酸</td><td>ギブコ</td><td> 25030 </td><td></td></tr><tr><td> EGF </td><td>インビトロジェン</td><td> PMG8041 </td><td> 20μlの単位にホルモンミックスとアリコート1mlの溶解。 </td></tr><tr><td> FGF </td><td>インビトロジェン</td><td> PHG0226 </td><td> 20μlの単位にホルモンミックスとアリコートを0.5mlで再構成する。 </td></tr><tr><td>ヘパリン</td><td>シグマ</td><td> H3149 </td><td></td></tr><tr><td>アポトランスフェリン</td><td> R＆Dシステムズ</td><td> 3188-AT </td><td> 0.1gを4ミリリットルのdH 2 0に溶解</td></tr><tr><td>プトレッシン</td><td>シグマ</td><td> P7505 </td><td>そうアポトランスフェリンに9.61 mgを溶解分離能</td></tr><tr><td>インスリン</td><td>シグマ</td><td> I5500 </td><td> 0.1NのHClを0.5mlに25mgを溶解するとdH 2 0の3.5ミリリットルに追加</td></tr><tr><td>セレン</td><td>シグマ</td><td> S9133 </td><td></td></tr><tr><td>プロゲステロン</td><td>シグマ</td><td> P6149 </td><td></td></tr><tr><td>標準解剖ツール</td><td>ファイン科学ツール</td><td></td><td></td></tr><tr><td>解剖顕微鏡</td><td>ツァイス</td><td> STEMI 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> 走電性商工会議所の準備 </td></tr><tr><td>正方形のガラスカバースライド</td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> 6N塩酸</td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td>高真空用グリース</td><td>ダウコーニング</td><td></td><td></td></tr><tr><td> 60ミリメートルペトリ皿</td><td>フィッシャー·サイエンティフィック</td><td> 0875713A </td><td></td></tr><tr><td>ポリエルリジン</td><td>シグマ</td><td> P4707 </td><td></td></tr><tr><td>マトリゲル</td><td> BD Biosciences社</td><td> 354234 </td><td> 150μlの単位に解凍し、アリコート</td></tr><tr><td> FBS </td><td>インビトロジェン</td><td> 10082139 </td><td>上記で示したように、NPCの分化を誘導する場合にのみ使用し、それ以外の場合は、SFM + EFH培養培地を使用</td></tr><tr><td>計数顕微鏡</td><td>オリンポス</td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> 細胞タイムラプスイメージングを生きる </td></tr><tr><td>銀線</td><td> Alfa Aesar社</td><td> 11434 </td><td></td></tr><tr><td>超高純度アガロース</td><td>インビトロジェン</td><td> 15510-027 </td><td></td></tr><tr><td>熱Inactivated FBS </td><td>シグマ</td><td> 16140071 </td><td></td></tr><tr><td> PVCチューブ</td><td>フィッシャー·サイエンティフィック</td><td> 80000006 </td><td> 3/32 &quot;内径x 5/32&quot; OD </td></tr><tr><td>漂白剤</td><td>クロロックス</td><td></td><td></td></tr><tr><td> 10ccの注射器</td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 18ゲージの針</td><td> BD </td><td> 305195 </td><td></td></tr><tr><td>ドレメルドリル</td><td>ドレメル</td><td>モデル750 </td><td></td></tr><tr><td>加湿、インキュベート室を備えた倒立顕微鏡</td><td>ツァイス</td><td> AXIOVERT-200M </td><td></td></tr><tr><td></td><td></td><td></td><td>レシピ<table border="1"><tbody><tr><td> アイテム </td><td> ボリューム </td></tr><tr><td> 2MのNaCl </td><td> 6.2ミリリットル</td></tr><tr><td>1MのKCl </td><td> 0.5ミリリットル</td></tr><tr><td> 1M MgCl 2を </td><td> 0.32ミリリットル</td></tr><tr><td> 155MMのNaHCO 3 </td><td> 16.9ミリリットル</td></tr><tr><td> 1Mグルコース</td><td> 1ミリリットル</td></tr><tr><td> 108 mMのCaCl 2 </td><td> 0.09256ミリリットル</td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td> 1ミリリットル</td></tr><tr><td>オートクレーブ処理水</td><td> 74ミリリットル</td></tr></tbody></table> 人工脳脊髄液 <table border="1"><tbody><tr><td> アイテム </td><td> 体積または質量 </td></tr><tr><td>人工脳脊髄液</td><td> 30ミリリットル</td></tr><tr><td>ウシ膵臓トリプシン</td><td> 40 mgの</td></tr><tr><td>羊は精巣ヒアルロニダーゼ</td><td> 22.8ミリグラム</td></tr><tr><td>キヌレン酸</td><td> 5mgを</td></tr></tbody></table> &lt;strong&gt;のトリプシン溶液<table border="1"><tbody><tr><td> アイテム </td><td> 体積または質量 </td></tr><tr><td> SFM </td><td> 15ミリリットル</td></tr><tr><td>オボムコイドトリプシンインヒビター</td><td> 10mgを</td></tr></tbody></table> トリプシン阻害剤溶液 <table border="1"><tbody><tr><td> アイテム </td><td> ボリューム </td></tr><tr><td>オートクレーブ処理水</td><td> 37ミリリットル</td></tr><tr><td> 10X DMEM/F12 </td><td> 10ミリリットル</td></tr><tr><td> 30％のグルコース</td><td> 2ミリリットル</td></tr><tr><td> 7.5パーセントのNaHCO 3 </td><td> 1.5ミリリットル</td></tr><tr><td> 1M HEPES </td><td> 0.5ミリリットル</td></tr><tr><td>トランスフェリン、プトレシンソリューション</td><td> 4ミリリットル</td></tr><tr><td> 25mgのインスリン溶液</td><td> 4ミリリットル</td></tr><tr><td>セレン</td><td> 100＆ムー;リットル</td></tr><tr><td>プロゲステロン</td><td> 100μlの</td></tr></tbody></table> ホルモンミックス （-20℃で100mlの合計、店舗） <table border="1"><tbody><tr><td> アイテム </td><td> ボリューム </td></tr><tr><td>オートクレーブ処理水</td><td> 37.5ミリリットル</td></tr><tr><td> 10X DMEM/F12（3:1） </td><td> 5ミリリットル</td></tr><tr><td> 30％のグルコース</td><td> 1ミリリットル</td></tr><tr><td> 7.5パーセントのNaHCO 3 </td><td> 0.75ミリリットル</td></tr><tr><td> 1M HEPES </td><td> 0.25ミリリットル</td></tr><tr><td>ホルモンミックス</td><td> 5ミリリットル</td></tr><tr><td> L-グルタミン酸</td><td> 0.5ミリリットル</td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td> 0.5ミリリットル</td></tr></tbody></table> 無血清培地EFH-SFM：0.5ミリリットルのFBSを追加したEGFの10μlに、FGFの10μl、およびヘパリンFBS-SFMの3.66μlを加える<taBLE国境= &quot;1&quot;&gt; <tbody><tr><td> アイテム </td><td> ボリューム </td></tr><tr><td>マトリゲル</td><td> 150μlの</td></tr><tr><td> SFM </td><td> 3.6ミリリットル</td></tr></tbody></table> マトリゲルソリューションマトリゲルのアリコートを氷の箱に入れて、SFMと混合する前に、粘性のある液体を形成するために4-5時間かけてゆっくりと解凍させなければならない。これは、マトリゲル基板の平滑な層の形成を確実にするでしょう。ゆっくりと解凍されていない場合は、得られた基板は、おそらく細胞遊走を阻害する、マトリゲルの塊を含んでいます。 <table border="1"><tbody><tr><td> アイテム </td><td> 体積または質量 </td></tr><tr><td>超高純度アガロース</td><td> 10ミリリットルのddH 2 0で300mgの</td></tr><tr><td> SFM 熱不活性化FBS </td><td> 8ミリリットル 2ミリリットル</td></tr></tbody></table> マトリゲル解決策は、15ccのファルコンチューブに2ミリリットル熱不活性化FBSでSFMの8ミリリットルを混ぜる。ミックスの三角フラスコ中で10ミリリットルのddH 2 0のアガロース、および30秒で、10秒間隔のために電子レンジでの加熱は、各10秒間隔の後に電子レンジから溶液を除去し、徹底的に混ぜて確保。最後の10秒電子レンジ期間の後、57℃の湯浴のSFM / FBS溶液とストアとアガロース溶液を混ぜる。 </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

哺乳類成体脳における神経幹細胞および前駆細胞の発見（以下総称して呼ばれる神経前駆細胞）（NPCは）神経再生戦略の開発のため、これらの細胞の多能性や増殖特性を利用することを目的とした研究の本体につながっている。そのような戦略の成功のための重要なステップは、外因性の移植後の病変部位に向かってNPCの動員であるかCNSの脳室周囲白質領域で発見された内因性の前駆体の応答を増強する。したがって、それは、推進導き、NPCの移行を強化するメカニズムを理解することが不可欠です。走電性として知られている現象 - 私たちの仕事は、NPCの移行を促進し、指示するために直流電界の利用（dcEFs）に焦点を当てています。内因性の生理的な電界が正常な発達と創傷修復中に細胞遊走のための重要な手がかりとして機能する。の薬理学的混乱アホロートル胚におけるトランス神経管電位は重度の発達奇形1を引き起こします。このDCEF 2-3の薬理強化や中断によって示されるように、創傷治癒のコンテキストでは、傷ついた角膜の修復速度が直接、損傷後生じる上皮創傷電位の大きさと相関している。我々は、外部から印加されたDCEFにさらされたときに成人上衣下NPCが試験管内で迅速かつ監督陰極移行を受けることを明らかにした。このプロトコルでは、高分解能、単一細胞レベルでディレク細胞体転座（マイグレーション）の長期観測のためのシンプルで効果的な走電性アッセイを作成するために私たちの研究室の手法について説明します。このアッセイは、トランスジェニックやノックアウトマウスでは、短鎖干渉RNA、または特定の受容体アゴニスト/アンタゴニストの使用を介して細胞の運動性にdCEFの伝達を制御するメカニズムを調査に適しているであろう。

キネマティック解析は、250 mVの/ミリメートルDCEFの存在下で、未分化なNPCが陰極に向かって非常に導かれ、急速な走電性（ 図5A、映画1）を示すことが明らかになった。 DCEFの非存在下では、細胞のランダムな動きが（ 図5B、ムービー2）が観察された。この電界強度では、未分化のNPCの&gt; 98％が撮像されているために全体の6〜8時間のために移行し、死細胞は移行され...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

このプロトコルでは、走電性の間に神経前駆細胞の転座に由来する成体脳のタイムラプスイメージングを可能にするために直流電界を印加することを許可するカスタムチャンバーを構築する方法を示します。

外部から印加された直流電界における神経前駆細胞の移行動態の解析のための走電性アッセイ

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Research

JoVE Journal

Neuroscience

12.4K ビュー.  University of Toronto. このプロトコルでは、走電性の間に神経前駆細胞の転座に由来する成体脳のタイムラプスイメージングを可能にするために直流電界を印加することを許可するカスタムチャンバーを構築する方法を示します。

ビデオ: A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神経コロニー形成細胞アッセイ：神経前駆細胞から善意の神経幹細胞を区別するアッセイ

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

神経科学

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

修正Zigmondチャンバーアッセイを使用してトランク神経冠細胞移動の解析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

クロス種の移植による神経堤の移行および分化の解析

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神経芽細胞アッセイ：フローサイトメトリーを用いて神経幹細胞の子孫を差別から神経前駆細胞の生成と濃縮のためのアッセイ

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

マウス腸の神経前駆細胞の定量細胞遊走アッセイ

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

皮質局所電界電位、心電図、筋電図、呼吸リズム自由行動ラットからの同時録音

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

電圧感受性染料を用いた脳スライスにおける広視野単一光子光学記録

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

胎児および成人神経幹細胞由来オリゴデンドロビテ前駆細胞培養の高含有スクリーニング分化と成熟解析

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...